Ion-Specific Self-Assembly of Hydrophobically Modified Polycation of

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Ion-Specific Self-Assembly of Hydrophobically Modified Polycation of Natural Origin Evgeniya V. Korchagina and Olga E. Philippova* Physics Department, Moscow State University, Moscow 119991, Russia S Supporting Information *

ABSTRACT: The effect of two monovalent salts differing in the anions (sodium acetate and sodium chloride) on intermolecular aggregation of fully protonated hydrophobically modified (HM) chitosan in dilute aqueous solutions was studied by light scattering. It was demonstrated that both salts promote the aggregation of similarly charged macromolecules. At the same time, strong ion specificity is observed: acetate ions induce the growth of the aggregates, whereas chloride ions increase the fraction of aggregated chains. The behavior observed was attributed to different mechanisms of interactions of these ions with HM chitosan. Acetate ions possessing higher affinity to chitosan involving electrostatic and hydrophobic interactions as well as hydrogen bonding screen more effectively the electrostatic repulsion between polycationic chains, which allows accommodating more macromolecules within one aggregate. On the other hand, more compact chloride ions seem to promote the formation of crystalline zones within the aggregates, thereby increasing the association energy in the system. This provides a higher fraction of associated chains, whereas keeping a small aggregation number, because large amount of macromolecules in one aggregate can hinder sterically a proper mutual arrangement of the chains necessary for the formation of crystallites.

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INTRODUCTION Chitosan, a linear polysaccharide composed of β(1 → 4) linked glucosamine and N-acetylglucosamine residues, is produced industrially by alkaline deacetylation of widely distributed natural polysaccharide chitin.1,2 Being a byproduct of marine food production, chitosan is a cheap polymer. At the same time, it possesses a unique set of properties: in addition to its excellent biocompatibility and complete biodegradability in combination with low toxicity,1−3 it demonstrates a number of different bioactivities (immunoadjuvant, antimicrobial, and others).4 Because of these properties, chitosan is widely used in medicine, biotechnology, etc.1 A special interest for these applications represents 100 nm aggregates of chitosan that are spontaneously formed in its dilute solutions.2,5−14 The self-aggregation is significantly enhanced in hydrophobically modified (HM) chitosan due to a t t r a c t iv e i n t e r a c t i o n s b e t w e e n it s h y d ro p h o b i c units.2,6,11,13,15−24 Aggregates of HM chitosan are quite prospective as drug delivery vehicles, particularly for hydrophobic drugs.3,22−24 Positive charge of the aggregates is expected to favor their transportation across cell membranes and ensure mucoadhesive and antimicrobial properties.25−27 In addition, these aggregates are promising as carriers of genetic materials in gene therapy19,24,28 since hydrophobic groups are known to enhance transfection efficiency.29 Taking into account that such applications suggest the presence of biological liquids, which always contain salts, it is important to understand the role of salt ions in the formation of aggregates. Theoretical considerations30,31 show that although © XXXX American Chemical Society

the aggregation is promoted by a large energy cost of contacts of hydrophobic groups with water, the size of aggregates is limited by Coulomb repulsion of similarly charged chains and by a loss of translational entropy of counterions which increase at the growing of the aggregates. Therefore, salt, which screens the electrostatic repulsion and decreases the losses in entropy of counterions, should favor the aggregation. Indeed, it was shown13 that at the increase of the amount of salt in solution (0.025−0.1 M) the weight fraction of aggregates gets bigger. At the same time, their aggregation number remains unchanged,13 which was attributed to the core−shell structure of the aggregates providing a low surface energy and strong attraction of associating groups in the core. Effective interactions between polyions in aqueous media depend crucially not only on the concentration of salt but also on the type of its ions. Ion-specific effects were first observed in the late 19th century, when Hofmeister demonstrated a selective “salting in” and “salting out” of protein by adding different salts to the solution.32 It is believed that the ion specificity relies on the peculiar electron cloud configuration33 determining the polarizability of ion and its interactions with solute and solvent. Now it is recognized that the specific ion effects influence many phenomena including the association of polyelectrolytes in aqueous medium.34−36 Some manifestations of ion specificity were already observed in the aggregation of Received: October 7, 2015 Revised: November 14, 2015

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DOI: 10.1021/acs.macromol.5b02213 Macromolecules XXXX, XXX, XXX−XXX

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ref 42. The degree of polymerization remained unchanged throughout this process. Two HM chitosan samples with 2 and 4 mol % of Ndodecyl-D-glucosamine units as determined by 1H NMR were prepared. They are further denoted as HMC-2% and HMC-4%. Sample Preparation. For the study of aggregation, 0.3 M CH3COOH aqueous solution containing different amounts of salt was used as a solvent. To prepare stock chitosan (or HM chitosan) solution (1.0−1.5 g/L), the solvent preliminarily filtered through a 0.22 μm filter was added dropwise to a weighed amount of polymer. After polymer dissolution, which takes several days, the solutions were passed through a 0.45 μm filter, and the concentration of polymer in the filtered solution was quantitatively determined by the ninhydrine method.43−45 It was demonstrated that the polymer loss as a result of filtration is less than 2%. To get chitosan (or HM chitosan) solutions with lower concentrations, filtered solvent was used to dilute the stock solution. The pH of 0.3 M CH3COOH solution was equal to 2.6 in the presence of 0.05 M NaCl and to 3.9 in the presence of 0.05 M CH3COONa. Taking into account that the pK0 of chitosan is 6.4− 6.5,46 in both solutions all amino groups of the polymer should be protonated. In such system, the average distance between the charged units along the chain is ca. 5.5 Å. It is much smaller than the estimated value13,47 of the Debye screening length rD equal to 13.6 and 19 Å at salt concentrations under study (0.05 and 0.025 M, respectively). Therefore, salt does not shield completely the electrostatic interactions in chitosan and HM chitosan solutions. Light Scattering Measurements. SLS and dynamic light scattering (DLS) experiments were carried out on an ALV/DLS/ SLS-5000 compact goniometer system at 25 °C with a helium−neon laser (632.8 nm) as a light source. The details of the treatment of light scattering data are presented in the Supporting Information. Refractive Index Increment Determination. The values of refractive index increment, dn/dc, at 632.8 nm were determined with an Optilab refractometer for HM chitosan solutions preliminarily degassed for removing air bubbles. Although these solutions always contain aggregates, their refractive indices scaled linearly with polymer concentration. The dn/dc values determined from the slope of these dependences are presented in Table 1. It is seen that they are the same in HM chitosan samples with different content of hydrophobic moieties, but they depend on the nature of added salt.

chitosan. In particular, an increased tendency to aggregation was found in NaCl solutions when compared to CH3COONa solutions.37 By molecular dynamics and quantum mechanics, it was shown that indeed chloride and acetate counterions can have different mechanisms of interaction with positively charged chitosan chain.38 The tendency of chitosan to aggregation can be even completely suppressed, if ammonium acetate is used as a salt,8,9 but it may not be always the case.39 As to the hydrophobic derivatives of chitosan, to the best of our knowledge, no study of the ion specific effects on their aggregation was performed up to now. As concerns any associating polyelectrolyte in general, it was never explored how the type of ions affects such important characteristics of the aggregation process like the number of macromolecules residing in one aggregate or the fraction of aggregated chains in the system. The present paper is aimed at examining the impact of the type of monovalent salt on the aggregation phenomenon in dilute aqueous solutions of HM chitosan, in particular, on such parameters like aggregation number and fraction of aggregated chains. For this purpose, two most widespread salts used for the preparation of chitosan solutions were chosen: sodium acetate and sodium chloride. The salts differ only in the type of anions. Although the value of charge of the anions is the same, their structure is quite different. Chloride is a compact spherical ion, whereas acetate possesses a hydrophobic group and an asymmetric structure with the negative charge concentrated on two oxygen atoms. Below we will show that these ions produce quite different impact on the aggregation behavior of HM chitosan, and we will discuss the possible reasons for the observed ion specificity.

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EXPERIMENTAL SECTION

Materials. Acetic acid (Fluka, 99.8%), sodium acetate (Fluka, 99.5%), sodium chloride (Helicon, 99.5%), and ammonium acetate (Helicon, 99.5%) were used as received. Water was distilled and deionized by a Milli-Q (Millipore) water purification system. Chitosan obtained by alkaline deacetylation of chitin extracted from crab shells was purified as described in details in ref 40. According to both potentiometry and 1H NMR data,41 the degree of acetylation of chitosan is equal to 0.05. The initial high molecular weight chitosan sample was depolymerized by acid hydrolysis and fractionated13 to get a sample with Mw = 75 000 g/mol (degree of polymerization 460). The value of Mw was determined by static light scattering (SLS) under conditions suppressing the self-aggregation, namely, (i) in 0.3 M CH3COOH/ 0.2 M CH3COONH4 solvent known to break the intermolecular Hbonds in chitosan8,9 and (ii) just after passing the chitosan solution through a 0.45 μm filter (to avoid the re-formation of aggregates disrupted in the course of filtration).13 HM chitosan samples with n-dodecyl side groups (Chart 1) were prepared by chemical modification of chitosan via reductive amination in homogeneous conditions according to the technique described in

Table 1. Refractive Index Increments of HM Chitosan Solutions in 0.3 M CH3COOH at Different Concentrations of Salt at 25 °C polymer sample

salt

Csalt (M)

HMC-2%

CH3COONa

0.025 0.05 0.025 0.05 0.025 0.05 0.025 0.05

NaCl HMC-4%

CH3COONa NaCl

dn/dc (mL/g) 0.187 0.185 0.200 0.195 0.187 0.185 0.200 0.195

± ± ± ± ± ± ± ±

0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005

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RESULTS AND DISCUSSION The aggregation behavior of polycationic chains of HM chitosan in dilute aqueous solutions containing 0.3 M CH3COOH was studied in the presence of two monovalent salts, NaCl and CH3COONa, differing by their anions. DLS data show that for all the samples under study the correlation functions g(1)(q,t) are bimodal (Figure 1):

Chart 1. Chemical Structure of HM Chitosan under Study (x = 0.05, z = 0.02 or 0.04)

⎛ t ⎞ ⎛ t ⎞ g(1)(q , t ) = A fast exp⎜ − ⎟ + A slow exp⎜ − ⎟ ⎝ τfast ⎠ ⎝ τslow ⎠ B

(1)

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Macromolecules Here Afast (Aslow) and τfast (τslow) are the amplitude and the relaxation time for the fast (or the slow) mode.

where D0 is the translational diffusion coefficient at infinite dilution and kD is the interaction parameter, which includes both thermodynamic and hydrodynamic contributions.51 To get the D0 values, the apparent diffusion coefficients were extrapolated to zero polymer concentration (Figures 3 and 4).

Figure 1. Field autocorrelation function g(1)(q,t) (1) and the distribution function of decay time A(t) (2) for 0.8 g/L aqueous solution of HM chitosan HMC-4% containing 0.3 M acetic acid and 0.025 M sodium acetate at scattering angle of θ = 90°. The fast peak corresponds to single coils, and the slow peak corresponds to the aggregates.

Figure 3. Dependence of the apparent diffusion coefficient of single macromolecules (fast mode) on the concentration of polymer in dilute aqueous solutions of HM chitosan HMC-2% in 0.3 M acetic acid containing 0.05 M of sodium chloride (1) and sodium acetate (2).

Figure 4. Dependence of the apparent diffusion coefficient of aggregates (slow mode) on the concentration of polymer in dilute aqueous solutions of HM chitosan HMC-2% in 0.3 M acetic acid containing 0.025 M of sodium chloride (1) and sodium acetate (2). Figure 2. Dependence of the relaxation rate on the square of scattering vector q2 for fast (1) and slow (2) diffusion modes for 0.8 g/L aqueous solution of HM chitosan HMC-4% containing 0.3 M acetic acid and 0.025 M sodium chloride.

From D0 values the hydrodynamic radii RH of the species were calculated using the Einstein−Stokes equation:52 RH =

For both relaxation modes, the dependencies of the relaxation rates Γ on the square of scattering vector q2 are linear and pass through the origin (Figure 2), which allows us to suggest that these modes are related to the translational diffusion of particles. As was shown earlier,11 the fast mode corresponds to individual macromolecules (unimers) and the slow mode to multichain aggregates. From the slope of Γ(q2) plots the apparent diffusion coefficients Dapp of the species were estimated. As a result of inter- and intrachain polymer−polymer interactions48 the values of Dapp are dependent on the concentration of polymer in solution. In the dilute regime, this dependence can be expressed as49,50 Dapp = D0(1 + kDc + ...)

kBT 6πD0ηs

(3)

where ηs is the solvent viscosity. The results obtained are summarized in Tables 2 and 3. Unimers. Let us first consider the data for nonaggregated macromolecules responsible for the fast mode. Figure 3 shows that for them the slope of the concentration dependencies of Dapp representing the interaction parameter kDuni is positive, indicating repulsive interchain and intrachain interactions.48 Lower kD values imply weaker polymer−polymer repulsion.51 From Table 2 it is seen that the enhancement of polymer hydrophobicity and the increase of salt concentration lead to smaller kDuni values; i.e., these factors diminish the repulsion between and within the chains. Note that the kDuni values in CH3COONa solutions are always much smaller than in NaCl solutions (Figure 3 and Table 2), indicative of weaker repulsion

(2) C

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the full length of n-dodecyl moiety should consist of ca. 40 ndodecyl groups. In HM chitosan under study the amount of C12 groups in one macromolecule is much smaller (9 for HMC-2% and 18 for HMC-4%), which renders their intramolecular aggregation quite unfavorable. Therefore, one can suggest two main reasons for the shrinking of HM chitosan macromolecules in salt solutions: screening of electrostatic repulsion between similarly charged units and decrease of the osmotic pressure exerted by the counterions located inside the polymer coils. The fact that in CH3COONa the hydrodynamic radii of unimers are much smaller than in NaCl can be explained by more effective screening of the charges on HM chitosan chains by acetate ions. Less extended conformation of chitosan chains in CH3COONa solution is consistent with smaller exponent a in the dependence of intrinsic viscosity [η] on the molecular weight M (Mark−Houwink−Kuhn−Sakurada relation [η] = KMa) equal to a = 0.76,37 in comparison with that in NaCl solution: a = 0.92−1.1.39 Thus, acetate counterions demonstrating a higher affinity to chitosan polyion provide more effective charge screening, which makes unimers more compact than in the presence of chloride counterions. Aggregates. Now let us consider the data for the aggregates (slow mode). From Figures 3 and 4 it is seen that in contrast to single chains, for aggregates the slope of the concentration dependence of the apparent diffusion coefficient is negative. It indicates that the interparticle interactions change from repulsion to attraction. Note that in this case the values of the interaction parameter kD are only slightly sensitive to the type of added salt being somewhat lower in acetate solutions. As to the hydrodynamic radii of aggregates, in both salts they are almost the same for HM chitosan HMC-2%, whereas for more hydrophobic polymer HMC-4% they are essentially larger in CH3COONa solution. This effect can be also explained by more effective screening of the charges on HM chitosan chains by acetate ions, which facilitates intermolecular aggregation of similarly charged HM chitosan polyions, if they contain a sufficient amount of associating n-dodecyl substituents. The same reason can explain the larger values of the gyration radii of aggregates (Table 3) in the presence of CH3COONa. As to the ratio (Rg/RH)agg, it lies in the range 0.44−0.62 (Table 3), which is even lower than for the hard sphere (0.778).53,54 As was demonstrated earlier,11 so small values of (Rg/RH)agg may indicate that the scattering objects have a dense core and loose shell, which makes their hydrodynamic radius much larger than the gyration radius. One can suggest that within this core−shell

Table 2. Diffusion Coefficients at Infinite Dilution and Interaction Parameters for HM Chitosan Solutions in 0.3 M CH3COOH Containing Different Salts Csalt (M)

salt CH3COONa NaCl

CH3COONa NaCl

0.025 0.05 0.025 0.05 0.025 0.05 0.025 0.05

D0uni × 10−7 (cm2/s)

D0agg × 10−8 (cm2/s)

kDagg (L/mol)

1.31 0.77 1.7 1.5

1.6 1.3 1.6 1.5

−0.50 −0.42 −0.38 −0.39

0.17 0.17 0.85 0.69

1.7 1.4 1.8 1.8

−0.35 −0.38 −0.33 −0.33

kDuni (L/mol)

HMC-2% 1.3 1.3 1.0 1.0 HMC-4% 1.8 1.8 1.3 1.3

in the presence of acetate ions. This can be due to more effective screening of the charges on HM chitosan chains by acetate ions. One may suggest several reasons for that. The first one is a stronger electrostatic interaction of CH3COO− ions with protonated amino groups providing resonance stabilization of −NH3+ ion between two oxygen atoms sharing the negative charge of carboxy group. The second reason is the hydrophobic interaction of acetate groups with polymer chains due to the presence of hydrophobic methyl group and asymmetric (surfactant-like) structure of the ion. The third reason is the formation of hydrogen bonds between acetate ions and both the amine site and the OH group on carbon atom 3 of glucosamine residue as it was demonstrated by computer modeling.38 As a result of these multiple interactions, the binding energy of acetate ions to protonated chitosan chains is much higher than for chloride ions, which was confirmed by quantum calculations.38 From Table 3 it is seen that in CH 3COONa the hydrodynamic radii of unimers are smaller than in NaCl, implying stronger contraction of polymer coils. As was demonstrated earlier,11,13 the hydrophobic units of HMC-2% and HMC-4% do not contribute to the compactization of single coils in salt solutions as the hydrodynamic radii of unimers of HM chitosan do not change with increasing content of hydrophobic units in polymer and are equal to those of unmodified sample. Responsive for the lack of intramolecular aggregation are rather high persistence length of chitosan1 and a small amount of hydrophobic side groups in one macromolecule. Indeed, one can easily estimate that a spherical hydrophobic microdomain with the radius of 15.4 Å equal to

Table 3. Radii of Unimers and Aggregates in Dilute Aqueous Solutions of HM Chitosan in 0.3 M CH3COOH Containing Different Salts Csalt (M)

CH3COONa

0.025 0.05 0.025 0.05

19 14 24 19

± ± ± ±

2 1 2 1

0.025 0.05 0.025 0.05

19 14 24 19

± ± ± ±

2 1 2 1

NaCl

CH3COONa NaCl

a

RH,unia (nm)

salt

RH,agga (nm) HMC-2% 155 ± 5 145 ± 5 158 ± 5 134 ± 5 HMC-4% 190 ± 5 180 ± 10 160 ± 5 134 ± 5

Rg,agg (nm)

(Rg/RH)agg

φagg × 10−4

82 90 72 71

± ± ± ±

2 2 1 1

0.53 0.62 0.44 0.53

14.5 14.2 8.5 9.0

85 102 75 72

± ± ± ±

2 2 2 2

0.45 0.57 0.47 0.54

13.0 10.6 8.4 9.2

The values are extrapolated to zero concentration of polymer. D

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Table 4. Apparent Weight-Average Molecular Weight Mw*, Second Virial Coefficient A2, Weight Fraction of Aggregates xagg, and Their Aggregation Numbers Nagg for Dilute Aqueous Solutions of Chitosan and HM Chitosan in 0.3 M CH3COOH Containing Different Salts

structure the hydrophobic fragments aggregating with each other are buried in the internal core, while most hydrophilic residues form a highly swollen shell exposed to water. The shell may contain many dangling chains formed by hydrophilic terminal fragments of HM chitosan macromolecules as well as some loops consisting of hydrophilic fragments from the middle part of polymer chains.11 From Table 3 it is seen that the (Rg/RH)agg values become higher with increasing salt concentration; i.e., the highly swollen shell shrinks more considerably than the core approaching the system to a homogeneous sphere. From the light scattering data the mean density of the aggregates, φagg, was estimated as described in the Supporting Information. It was shown (Table 3) that it is much bigger in CH3COONa when compared to NaCl, which may be due to more effective screening of electrostatic repulsion by acetate ions. Now let us consider SLS data. Figure 5 shows typical SLS results in the form of a Zimm plot. Linear angular dependences

salt

Mw* (kg/mol)

A2 × 104 (cm3 mol/g2)

xagg

Nagg

chitosan CH3COONa NaCl

CH3COONa NaCl

CH3COONa NaCl

0.025 0.05 0.025 0.05 0.025 0.05 0.025 0.05 0.025 0.05 0.025 0.05

120 150 145 190 HMC-2% 350 600 310 540 HMC-4% 400 800 530 700

8.0 5.0 12.0 10.0

0.06 0.08 0.10 0.16

13 15 10 11

± ± ± ±

1 2 1 1

5.0 2.0 10.0 2.1

0.08 0.15 0.17 0.31

47 48 19 19

± ± ± ±

6 5 2 3

2.0 1.5 7.0 1.6

0.11 0.20 0.33 0.38

50 49 20 20

± ± ± ±

8 3 2 3

To get Magg values, Muni and Mw* data were taken from SLS results, whereas xagg data were determined from DLS measurements as described elsewhere.11 The aggregation numbers Nagg were estimated as the ratio Magg/Muni. The values of Nagg and xagg are summarized in Table 4. It is seen that they depend drastically on the type of added salt. Surprisingly, the effects of both salts on Nagg and xagg values are quite opposite: in CH3COONa the aggregation numbers are much larger, whereas in NaCl the fraction of aggregated chains is higher. In other words, in sodium acetate solutions we have a small number of big aggregates, whereas in sodium chloride solutions a large number of tiny aggregates, besides in the last case the total amount of aggregated chains is much higher. Larger Nagg values in CH3COONa solutions may be due to more efficient screening of electrostatic repulsion by acetate ions, which allows accommodating more similarly charged chains in one aggregate. It should be noted that the effect of CH3COONa on the aggregation number is much more pronounced for HM chitosan than for its unmodified precursor (Table 4). Indeed, in CH3COONa the Nagg values of chitosan are only slightly higher than in NaCl, whereas for HM chitosan a 2.5-fold difference is observed (Table 4). Now let us consider the fraction of aggregated chains xagg. From Table 4 it is seen that it is much larger in NaCl solution. This may result from stronger attraction between HM chitosan macromolecules caused by some additional contribution to the association energy, which may be due, in particular, to the formation of crystallites in junction zones linking different chains together. The crystallization of junction zones in the aggregates of chitosan was already discussed in some papers.12,59−61 It was suggested that the crystallites are formed by stereoregular fragments of different chains arranged parallel to each other. Such crystallites are highly sensitive to counterions.59 For instance, in paper59 by isothermal calorimetric measurements of the concentration dependences of the enthalpy of dilution of chitosan solutions, it was demonstrated that the crystalline regions are formed by chitosan chloride, whereas no crystallites were detected in chitosan acetate. The fact that chitosan chloride is prone to

Figure 5. Zimm plot for aqueous solutions of HM chitosan HMC-2% in the range of concentrations 0.25−0.9 g/L in 0.3 M acetic acid containing 0.025 M sodium chloride.

without curvature at any concentration of HM chitosan indicate a “closed” model of association55 (i.e., discontinuous formation of rather monodisperse aggregates56). The values of the apparent weight-average molecular weight Mw*, the apparent z-average radius of gyration Rg*, and the second virial coefficient A2 determined from the Zimm plots are presented in Table 4. It is seen that in both salts the values of A2 are positive revealing the stability of the solutions. In CH3COONa the second virial coefficient A2 is smaller, which may be due to more compact conformations of HM chitosan chains as a result of stronger screening of electrostatic repulsion by acetate ions. From Table 4 it is seen that the apparent weight-average molecular weight Mw* is much bigger than for single macromolecules indicating the presence of intermolecular aggregates. SLS results were combined with DLS data to recover the molecular weight of these aggregates Magg by using the equation57,58 M w * = (1 − xagg)M uni + xaggMagg

Csalt (M)

(4)

where Muni is the weight-average molecular weight of individual nonaggregated macromolecules and xagg is the weight fraction of aggregates. E

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form crystallites as opposed to chitosan acetate was attributed to the compactness of chloride counterions. We suggest that the ability of chitosan chloride to crystallization may be responsible for the increased number of chitosan macromolecules involved in the aggregation in NaCl solution. Surprisingly, the additional contribution to the association energy provided by crystallization does not lead to the growth of aggregates in this case. This may be due to the fact that large content of macromolecules in the aggregate can hinder sterically a proper mutual arrangement of the chains necessary for the formation of crystallites. Note that the impact of the type of salt on xagg values is the same for chitosan and HM chitosan as in both cases xagg decreases by a factor of 2 when passing from NaCl to CH3COONa (Table 4). Therefore, the salt effect on the fraction of aggregated chains xagg is independent of the hydrophobicity of polymer, whereas the salt effect on the aggregation number Nagg increases at the hydrophobic modification of macromolecules. This may indicate a significant role of nonmodified chitosan units in the specific ion effect on the fraction of aggregated chains. Thus, the present paper demonstrates that the mechanisms of the enhancement of the aggregation of HM chitosan in the presence of acetate and chloride salts are quite different: acetate ions induce the growth of the aggregates, whereas chloride ions promote increasing fraction of aggregated chains. To the best of our knowledge, such an opposite effect of the type of ions on two main characteristics of the aggregation process has not been observed before in any associating polyelectrolyte.

E.V.K.: Department of Chemistry and Faculty of Pharmacy, University of Montreal, CP 6128 Succursale Centre Ville, Montreal, QC H3C 3J7, Canada. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of the Russian Foundation for Basic Research (project 14-03-00934) is gratefully acknowledged. The authors express their gratitude to Dr. I. V. Blagodatskikh for fruitful discussions.

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CONCLUSIONS The intermolecular aggregation of positively charged HM chitosan macromolecules in water medium in the presence of two different monovalent salts (sodium acetate and sodium chloride) was studied by dynamic and static light scattering techniques. Both salts were shown to enhance the aggregation. At the same time, spectacular ion specific effects were observed: the larger aggregates were formed in the presence of acetate ions, whereas chloride ions induce the involvement of bigger amount of polymeric chains in the aggregation. These differences were attributed to the peculiar structure of acetate ions providing them higher efficiency in screening electrostatic repulsion and to small size of chloride ions favoring close packing of chitosan chains with the formation of crystalline zones. The specific ion effects observed here are of obvious importance for numerous practical applications of saltcontaining aqueous solutions of chitosan and its hydrophobic derivatives. Moreover, these effects may be helpful for general fundamental understanding of the aggregation phenomena in any associating polyelectrolyte in the presence of salt.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b02213. Treatment of light scattering data, determination of specific volume (PDF)

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DOI: 10.1021/acs.macromol.5b02213 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.5b02213 Macromolecules XXXX, XXX, XXX−XXX